Method of improving yeast



' s.) -u A an. anti Slluflul" tubers The present application relates to subject mat- I Patented June 30, 1942 UNITED STATES PATENT OFFICE METHOD OF IMPROVING YEAST Lee M. Christensen, Atchison, Rana, assignor to The Chemical Foundation, Incorporated; a corporation of Delaware No Drawing. Application June 25, 1931, Serial No. 150,417

1 (Balm.

This invention relates to the production of alcohol by fermentation, more particularly to the production of ethyl alcohol by fermentation of the Jerusalem artichoke (Helianthus tuberosus) tains a relatively large percentage of polysaccharides, largely in the form of polylevulosans, and

some non-sugars.

The availability of this raw material for the production of alcohol has been recognized and processes involving its use have been carried out.

In the past, the methods employed haveinvolved a typical two-phase fermentation process. The first phase comprised the preliminary hydrolysis of the polysaccharides to simple 'fermentable sugars, by means of mineral acids or specific enzymes. The second phase consisted in the fermentation of these sugars. together withethe monosaccharides originally presennby means of typical yeasts, for the purpose of producing ethyl alcohol. If. such diffusion liquor were diorder ultimately to obtain alcohol from the polysaccharides; it was considered necessary to utiline this preliminary separate step to insure hydrolysis of the polysaccharides to the simpler sugars; Y

It has now been found that certain special strains of yeast may be developed which possess the ability to convert the polysaccharides, as well as the monosaccharides, contained in artichokediflusion liquor to'alcohol. As explained in the earlier application referred to, when such special yeast strains are contacted with unhydrolyzed diffusion liquor under suitable conditions,

conversions of from 90 to 94 per cent. of the car-.

bohydrate content to alcohol are obtained. Such novel strains therefore permit a single step conversion in which the polysaccharides, as well as *rectly sabjeciedntonislmentation, wi lthgya 7 the monosaccharides, are fermented. The manlfold advantages attained by effecting in this one step, that which heretofore required two distinct operations, will quickly be appreclated by those skilled in the art. p

This useful and specific function may be de-, veloped in certain yeastsin a manner more fully to be described so as to produce, in ell'ect; novel yeast strains ortypes.

An object of the present invention, therefore, is to improve the alcohol fermentation of tuber diffusion liquor by effecting such fermentation with new yeast strains.

Another objective of the invention is to pro-, duce improved yeast strains which are particularly efficacious in the conversion of carbohy:

drates to alcohol.

With these and" other "eduall'il important and I related objects in view, the invention comprehends the concept of generating improved yeast strains which are characterized by the ability to substantially completely ferment the carbohydrate content of Jerusalem artichokes and similar tubers.

The novel yeast strains contemplated herein may enerally be considered as bottom r inter mediate fermenting yeastsotthe geniissaccharo nycestowhichTby means of a special devel opment, selection, or acclimatization, is imparted the described additional, specific function. Since such yeasts are peculiarly efficacious in the treatnflent of Jerusalemartichokes, they may, for the sake of a term, be designated as Saccharomyces helianthus. The developed strains, as explained, may be used to ferment the diffusion liquor of, or a mash made from, artichoke tubers under either aerobic or anaerobic conditions to substantially completely convert the carbohydrate content.

The yeast which formed the bases for the new strains, are readily available and are found, for example, on grains, such as wheat and corn; on fruits, such as grapes and apples, and in the soil.

In producing the new strains, it is found that the Jerusalem artichoke liquor presents an excellent culture medium. This liquor may be produced by diffusing sliced tubers with warm water at a temperature of about to C. Such liquor usually contains about v12 to 15 or more per 'cent.'of total solids. It has been found advantageous to concentrate such liquor to, a syrup in which form it may bestored. Whenrequired, for the production of new yeast strains, the syrup may be withdrawn from storage and more.

When it is desired to propagate the yeast, such sterilized diffusion liquor is diluted so as to adjust the total carbohydrate content to approximately 5 per cent, The hydrogen ion concentration is preferably adjusted to a pH of 4.5 to 5. A series of the cultures is then made by inoculating tubes of such diffusion liquor with a small amount of the grain, fruit, soil, or other yeast source, and the culture is allowed to ferment with this type of treatment, it will be found that from 92 to 95 per cent. of the total carbohydrates of the artichoke tubers is converted to ethyl alcohol and carbon dioxide.

5 At the termination of the fermentation step,

for a period of from 6 to 7 or more days. Durin; such fermentation, thetemperature is preferably maintained at about 30 C. I

From this initial series'of'such cultures, those which show the most vigorous fermentation and largest productionof ethyl alcohol are chosen and are then utilized to inoculate additional quantities of the original culture medium. This process of selective inoculation is continued for from 5 to or more transfer cycles, during which the inoculation period is successively di the fermented diffusion liquor or mash may be treated by distillation methods employed in the art to produce aqueous or anhydrous alcohol.

It is particularly to be observed that the pres- 0 cut method of treatment of yeasts, to impart to them the capability of directly coverting the polysaccharides, is carefully to be distinguished from prior suggested methods of acclimatization. It has been known in the past that if certain yeast 5 which inherently possesses the ability to convert certain monosaccharides are acclimatized in such monosaccharides, this ability is considerably enhanced. The present method, as will be appreciated, involves the acclimatization of the initial yeast in a heterogeneous culture so as to fdevelwthmabilityio-cenvert polysaccharides.

In other words, the present method induces a new and specific function in the yeast, rather than an increaseor enhancement of a preex- 5 isting, inherent function.

The following characteristics will serve to more minished from the criwMtoT hei-ein:

days to abnul'aldaysa lized to make plates by employing a solid medium comprising about 1.5 per cent. of agar agar and the diffusion liquor from the artichoke tubers such that the final medium contains about 5 per cent of total carbohydrates. By this procedure, a substantially pure culture of the de- I suitable single'cell method.

Theultimate cultures obtained are then uti- Morphologicah-Yeast grown in beer wort.

Size of vegetation cells: 4.5to 8 microns by 3 to 6 microns Shape-of vegeta- 5 tion cells:

Method of multiplication: Mycelium: Spore fermenta- -0val Budding None The novel strains thus produced cai'rtherrbe io fl m directly employed for the production of ethyl alcohol and carbon dioxide from the diffusion liquor of artichoke tubers or from a mash made by maceration and cooking of such tubers. The

diffusion liquor may be producedin the usual manner by diffusing the tubers with water at 80 3., more or less. "If desired, the diffusion liquor 'may be evamrated to a syrup and stored, dilut- (On carrot infusion agar) l to 4 spores per ascus, 0.7 to 1.0 microns diameter, nearly round.

Carbohydrate fermentation (in yeast water meing to about 20 per cent carbohydrate content I before inoculation. k 4

In carrying out the fermentation, it is preferred to develop the inoculum in artichoke diffusion liquor. However, this is notessential, as it has been found that saccharifieMain mash or molasses may be employed. In any event, in the fermentation, the amount of. the

inoculum tothe diflusion liquor 'or mash pref-- erably should be adjusted to between 3 to 10 per cent. It is found that the best results are secured by concentration of the total carbohy drates in the fermentation adjusted to between 12 and 20 percent.

During the fermentation step, the hydrogen ,ion concentration should be established at between pH 4.5 to 5, and the temperature maintained at approximately 30 C., although this temperature may be allowed to rise to 42 C. as the fermentation proceeds. In these circumstances, it is found that in approximately 2 days, substantially complete conversion is effected. It will be found advisable to aerate the system during the first four to six'hours after inoculation, although such aeration is not essential. In any event, aeration should not be continued after about eight hours of fermentation.

dium). 1. Insulin- 2. Starch- 3. Sucrose+ 4. lviaitose+ 5. Lactose- 6. Ievulose+ 7. Glucose+ ,8. Mannose-i- 9. Dextrose+ 10. Polylevulosans of Helianthas tuberosusand S. 'anamensz's, which are effective for the stated purpose,-namely, the direct conversion of the polylevulosans'to alcohol and carbon dioxide. It is believed that in this case the operation is one of selection.

Intermediate -or bottom Similarly, schizosaccharomgces pombe and capable of directly fermenting the polysaccharides contained in Jerusalem artichokes, which comprises, preparing a medium containing such polysaccharides, adjusting the hydrogen ion concentration of the medium topH 4.5 to 5; inoculating the medium with distiller-s yeast and incubating at a temperature of substantially 30 C. for a period of approximately 5 to 7 days;

then successively inoculating a similar medium with the yeast propagated in the preceding incubation, while successively diminishing the time of incubation.

\ LEO M. CHRISTENSEN. 

